The present invention relates to the administration of an endogenous hormone peptide, more particularly, to the administration of an endogenous hormone peptide with gene-based material to enhance feed efficiency and growth rate of livestock.
There have been many approaches to stimulate the growth rate of livestock and to enhance feed conversion efficiencies. These include the use of antibiotics, chemicals, and the use of biological compounds such as native or recombinant growth hormones (GH) or growth hormone releasing factors (GHRF). Many of these approaches either have side effects or are too expensive to implement in actual farms or ranches.
Although antibiotic supplements have been routinely used to enhance growth performance and feed conversion in the past, their benefits are now diminishing due to improvements in modern farm management and the potential danger of spreading antibiotic resistance to other animals including man.
The earliest attempts in biological enhancement used injections of pituitary extracts, from which the pituitary GH was later isolated and purified.
GH produces a variety of effects on body tissues, ultimately leading to an increase in growth rate and weight gain. In vivo, GH stimulates the synthesis of proteins, the breakdown of fats, and epiphyseal growth. These effects are mediated via the up-regulation of somatomedins, for example insulin-like growth factor I (IGF-I). The production and release of pituitary GH is under the control of two hypothalamic hormone peptides. Inhibition by somatostatin and stimulation by GHRF regulates the plasma GH levels. As animals age, there are deleterious changes in the GHRFxe2x86x92GHxe2x86x92IGF-I axis. Hence in older animals including man, GH production rate decreases, and the IGF-I response to GH and GHRF diminishes. These lead to osteoporosis, decrease of lean muscle, and increasing truncal fat deposits. Therefore pituitary extracted GH has been used to supplement the natural decline in GH and enhance growth. However, such a treatment suffers from the risks of adventitious pathogen transmission. In addition, the major drawback is the high costs involved. Since GH is a protein, it is easily digested by intestinal enzymes and must be administered by injection. Furthermore, GH has to be given daily due to its short serum half-life. This pushes up the cost of treatment, making it feasible only for human therapy.
The development of recombinant DNA technology and the use of microbes such as E. coli to produce mammalian GHs meant that homogenous GH preparations could be produced in large quantities for treatment, thereby reducing the costs. Additionally, recent advances in protein stabilization by engineering substitutions in the primary structure and development of slow release formulations have reduced the frequency of dosing to weekly or bi-weekly. As the costs of treatment lower, it can be used commercially to boost agricultural production. For instance, recombinant bovine GH is currently being used to boost milk production in a third of the United States of America dairy herd.
Treatment of pigs with porcine GH was shown to enhance growth rate, increase carcass protein whilst reducing the proportion of fat. The benefits of porcine GH on pig""s growth performance and the safety and economic aspects involved have been reviewed by Palmer J. Holden (Porcine somatotropin (pST), Biotechnology Information Series (Bio-4), Iowa State University, 1993). Summarizing data from twenty studies, it was reported that porcine GH injected pigs grow 15% faster whilst consuming 21% less feed, with more muscle protein and reduced backfat. However, these gains in productivity are made at substantial costs. Firstly, because of its short serum half-life and rapid clearance from the bloodstream, GH must be injected daily to be effective. Hence pigs must be given up to 4 mg of porcine GH protein per day for the one or two month duration of the finishing period to sustain plasma GH levels and improve growth performance. Thus each pig may eventually require as much as 400 mg of GH. Secondly, high doses may be toxic and have been shown to cause some adverse side effects such as breathing difficulties, as described in the U.S. Pat. No. 5,134,120. Moreover, the treatment regiment so far described in the literature is too labor intensive and traumatic to the animal. Even though U.S. Pat. Nos. 5,015,626 and 5,134,120 describe the use of porcine GH to improve meat quality and enhance weight gain respectively, the current technology in GH peptide-based growth promotants are yet to be economical to apply to commercial farming.
The discovery of the growth hormone releasing factors (GHRF) promised to provide a better method of growth enhancement. GHRF is a peptide hormone secreted by the hypothalamus and specifically stimulates the synthesis and release of GH by the somatotroph cells of the anterior pituitary gland. By stimulating the endogenous production of GH, the effective dose required is much smaller than that of GH, thus providing a more physiologic mode of treatment at potentially lower costs. Nevertheless, to the best of our knowledge, there appears to be still no satisfactory and cost effective treatment for livestock. For example, the use of human GHRF protein injections twice daily to stimulate the growth of sheep was described in EP Pat. No. 0,289,186. However, prolonged injections of the human GHRF induce immune response in sheep with neutralizing antibody formation against the human protein. This lowers the efficiency of such a treatment, and the labor and material costs render this approach uneconomical. Injections of GHRF peptide have been used to produce a more sustained stimulation of production of GH in the pig. Although some trials have shown success in producing a gain in body weight and lower carcass fat content, the same problem of rapid clearance means frequent doses are required. Therefore a new approach to supplement GH levels for growth enhancement is required.
Since it was first demonstrated that intramuscular-injected plasmid DNA vectors are taken up by the skeletal muscle cells and expressed for many months in vivo, skeletal muscle based gene therapy has held great promise for the systemic delivery of therapeutic proteins. This skeletal muscle production of recombinant proteins which can be secreted into the circulation system to reach distant target sites ideally suites the treatment of diseases arising from serum protein deficiencies. Plasmid vectors, although much less efficient in protein expression than viral based vectors, have the advantages of being less likely to be immunogenic and not integrated into the host genome. Therefore, they are generally thought to be safer. On this basis plasmid vectors best suit situations where the therapeutic protein is effective at low concentrations. Thus, injection of plasmid vectors expressing the gene for GHRF may be an ideal method to apply a chronic supplement of GHRF at an effective dose sufficient to enhance growth performance of animals. Furthermore, persistent GHRF production should drastically reduce the frequency of treatment, hence lowering the costs to an economically viable level.
Recently, it was shown that a plasmid containing human GHRF (hGHRF) cDNA under the control of the chicken minimal skeletal xcex1-actin promoter is able to produce an elevation in plasma GH levels when injected into the mouse muscle (Draghia-Akli et al, Nature Biotechnology, 15, p1285-1289). Furthermore, growth enhancement of approximately 15% was reported at 3 weeks post injection. However, their methods elicited an immune response as evidenced by the increasing serum levels of neutralizing anti-hGHRF antibodies at 21 and 28 days after hGHRF gene injection. It has been shown that the limiting factor in the level of in situ gene expression from injected plasmids is the extremely low degree of uptake of plasmid DNA by the muscle fibers. To improve the level of hGHRF expression, Draghia-Akli and co-workers first injected bupivacaine, a well known myotoxic substance, before injecting the plasmid DNA into the regenerating muscle. Although this has been shown to improve the level of expression by increasing plasmid uptake in the muscle fibers, it is neither a desirable nor an acceptable technique to apply in farm animals reared for human consumption.
It is therefore an object of this invention to provide a cost-effective approach to stimulate the growth rate of animals, particularly livestock animals, and to enhance feed conversion efficiencies through the administration of an endogenous hormone peptide GHRF. As a minimum, it is an object of the present invention to provide the public with a useful choice.
Accordingly, there is provided a method of enhancing feed efficiencies and/or growth rates and/or reducing fat accumulation of animals comprising administration of endogenous hormone peptide GHRF by an exogenous gene sequence to stimulate the production of GHRF, and said exogenous gene sequence comprises a DNA sequence encoding a promoter for gene expression, a complementary DNA (cDNA) sequence encoding a GHRF signal peptide, and a cDNA sequence encoding GHRF.
Among the particularly preferred embodiments of this invention are variants of the promoter being an actin promoter. In accordance with certain preferred embodiments of this invention, the actin promoter is a skeletal actin promoter. The DNA sequence encoding a skeletal actin promoter particularly prefers comprising a full set of DNA sequence of the skeletal actin promoter.
In accordance with another preferred embodiments of this invention, the exogenous gene sequence further includes a DNA sequence encoding the three prime (3xe2x80x2) untranslated polyadenylation signal-containing region of the gene of the promoter or GHRF.
In accordance with yet another preferred embodiments of this invention, the exogenous gene sequence further includes a DNA sequence encoding a gene for antibiotic resistance.
The GHRF and its signal peptide encoded by said cDNA sequences can be the corresponding natural or recombinant or synthetic, or biologically active fragments or their analogues with similar activities. It is a particularly preferred embodiment of this invention that the DNA sequence of the promoter, the cDNA sequence encoding a GHRF signal peptide, and the cDNA sequence encoding GHRF are species specific.
The said exogenous means to stimulate the production of GHRF being a DNA sequence is preferably mixed with a suitable carrier to be administered subcutaneouly. In accordance with certain preferred embodiments of this invention, the exogenous DNA sequence is administered via intramuscular route.
The said exogenous means to stimulate the production of GHRF being a DNA sequence given per animal is preferably between the range of 1 to 100 xcexcg per kg body weight of the animal.
It is another aspect of this invention to provide an exogenous gene sequence to simulate the endogenous production of GHRF to enhance feed efficiencies and/or growth rates and/or reduce fat accumulation of animals, wherein the exogenous gene sequence comprise a DNA sequence encoding an promoter for gene expression, a cDNA sequence encoding a GHRF signal peptide, and a complementary DNA (cDNA) sequence encoding GHRF.
It is yet another aspect of this invention to provide a method of manufacturing a medicament to enhance the feed efficiencies and/or growth rates and/or reduce fat accumulation of animals comprises providing a DNA sequence encoding a promoter for gene expression, joining a cDNA sequence encoding a GHRF signal peptide, and joining a cDNA sequence encoding GHRF to form a gene sequence and mixing said peptide with a suitable carrier.
Other objects, features, advantages, and aspects of the present invention will become apparent to those skilled from the following description. It should be understood, however, that the following description and the specific examples, while indication preferred embodiments of the invention, are given by way of illustration only. Various changes and modifications within the spirit and scope of the disclosed invention will become readily apparent to those skilled in the art from reading the following description and from reading the other parts of the present disclosure.